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RNA-Mediated Trans-Activation of Transcription from a Viral RNA

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Science  07 Aug 1998:
Vol. 281, Issue 5378, pp. 829-832
DOI: 10.1126/science.281.5378.829

Abstract

The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.

RNA performs many of the functions that were once thought to be restricted to proteins. RNA molecules perform various enzymatic reactions in addition to catalyzing peptide bond formation (1). Given this diversity of functions, it is not surprising that gene expression can be regulated posttranscriptionally by the structure or stability of an mRNA (2). Noncoding RNAs as well as the 3′ untranslated regions (3′ UTRs) of cellular mRNAs function as trans-acting regulators of cell division and differentiation (3). In the nematodeCaenorhabditis elegans, the small noncoding lin-4 RNAs alter the stability or translatability (or both) of lin-14 mRNAs by interacting with their 3′ UTRs (4). However, RNA-mediated regulation of transcription from an RNA molecule has not been observed.

Red clover necrotic mosaic Dianthovirus (RCNMV) contains two RNA components, a polycistronic RNA-1, which encodes the viral polymerase and capsid protein (CP), and RNA-2, which encodes the viral movement protein (MP) required for the cell-to-cell spread of infection (5) (Fig. 1, A and B). CP is translated from a subgenomic RNA (sgRNA) transcribed from RNA-1 because the CP open reading frame (ORF) on RNA-1 is inaccessible to the eukaryotic translational machinery (6). RNA-1 alone is infectious in plant protoplasts (5) and can produce a local infection on the inoculated leaves of transgenicNicotiana benthamiana plants expressing the RCNMV MP (MP+ plants) (7). However, CP synthesis and subsequent systemic infection are observed only when RNA-1 is co-inoculated with wild-type RNA-2 or mutant RNA-2 transcripts that are incapable of MP synthesis (7).

Figure 1

Genome organization of RCNMV and TBSV. (A) Schematic diagram of RCNMV RNA-1, CP sgRNA, and R1sGFP. ORFs for p27 (polymerase component), the −1 ribosomal frameshift (FS) product p88, and the CP are labeled. R1sGFP is a construct in which the CP is replaced with the sGFP ORF (shaded box). sGFP is expressed from this modified genomic RNA-1 only when a subgenomic RNA is generated. (B) RCNMV RNA-2 and RNA-2 derivatives. R2ΔATG contains a deletion of the MP start codon and does not produce functional MP. R2GFP is a construct in which the MP is replaced with the wild-type GFP ORF (shaded box). R2GΔH is a construct derived from R2GFP in which 300 3′-terminal nucleotides of the GFP sequence are replaced by 324 3′-terminal nucleotides from the MP ORF. R2GΔHX is a construct derived from R2GΔH with a 120-nt deletion of the 3′-terminal MP ORF sequence. The length of transcripts in nucleotides is indicated to the right of the genomes. The nucleotide positions of the start and stop codons for the MP ORF are indicated above RNA-2. The position of the 34-nt trans-activator sequence located between nucleotides 756 and 789 is indicated by the asterisk. (C) Map of the wild-type TBSV genome, TBSV expression vector pHST2, and the CP sgRNA it produces, which was engineered to accept and express foreign genes. The deleted region of TBSV CP in pHST2 is indicated by the dotted line. RCNMV RNA-2 sequences are inserted 55 nt from the 5′-terminus of the sgRNA.

To investigate the requirement for RNA-2 in sgRNA synthesis, we engineered an RCNMV RNA-1 cDNA clone, from which infectious transcripts can be synthesized (8), to express a synthetic mutant form of the Aequorea victoria green fluorescent protein (sGFP) (9) in place of the CP (clone R1sGFP) (10) (Fig. 1A). Inoculation of R1sGFP transcripts onto MP+ plants resulted in the accumulation of sGFP only when wild-type or mutant RNA-2 transcripts were also present in the inoculum. RNA-2 transcripts from the related Dianthovirusescarnation ringspot virus (CRSV) (11, 12) and sweet clover necrotic mosaic virus (SCNMV) (13) also induced sGFP expression. These findings suggest that a conserved region of RNA-2 is required to elicit efficient transcription of sgRNA from RNA-1.

A chimeric RNA-2 clone with the wild-type GFP ORF in place of the MP ORF (clone R2GFP) (14) (Fig. 1B) was unable to replicate on MP+ plants when co-inoculated with wild-type RNA-1 transcripts. Replacement of the 3′-terminal 300 nucleotides (nt) from the GFP insert with the 3′-terminal 324 nt from the MP ORF (clone R2GΔH) (14) (Fig. 1B) restored RNA-2 replication. This RNA-2 construct was also able to elicit sGFP expression when co-inoculated with R1sGFP transcripts, suggesting that the 324-nt region contained cis-acting elements required for RNA-2 replication along with trans-acting sequences required for eliciting RNA-1 sgRNA synthesis. Deletion of the 3′-terminal 120 nt of the MP ORF from clone R2GΔH (clone R2GΔHX) (14) abolished replication of this RNA-2 construct, so we turned to another strategy for delimiting the trans-acting sequences.

To uncouple the trans-acting sequences required for RCNMV sgRNA expression from the cis-acting sequences required for RNA-2 replication, we used a heterologous RNA viral vector. An infectious cDNA clone of the monopartite tomato bushy stunt virus (TBSV) was previously engineered to allow transcription of foreign sequences from the CP subgenomic promoter (15) (Fig. 1C). The 324-nt fragment from RCNMV RNA-2, having the trans-activator activity, was cloned into the TBSV vector in both the genomic- and complementary-sense orientation (16). Transcripts of these constructs were co-inoculated with R1sGFP transcripts ontoN. benthamiana and Nicotiana clevelandii plants. sGFP expression was induced only when the RNA-2 insert was present and in the RCNMV genomic-sense orientation (Table 1).

Table 1

Expression of sGFP from RCNMV R1sGFP depends on RNA-2 sequences. R1sGFP was co-inoculated with transcripts from the TBSV replicon containing segments from RCNMV RNA-2 in order to delimit the minimal trans-activator element. See Fig. 1B for nucleotide positions on RNA-2. Complementary sequence is indicated by (−). Primer sequences used for PCR and color images of GFP expression can be viewed atwww.sciencemag.org/feature/data/981282.shl

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A smaller 209-nt RCNMV RNA-2 insert (representing the sequence present in clone R2GΔHX) (16) in the TBSV vector also induced sGFP expression when co-inoculated with R1sGFP transcripts. CP was expressed when this TBSV construct was co-inoculated with wild-type RCNMV RNA-1 transcripts (Fig. 2A). This expression suggests that the trans-activation effect is not gene-specific and that sGFP is a valid reporter to identify and delimit the cis- and trans-acting signals for CP sgRNA synthesis.

Figure 2

Analysis of RCNMV RNA-1 sgRNA synthesis and CP gene expression with RCNMV RNA-2 sequences expressed from the TBSV replicon. (A) Immunoblot analysis of CP production. Total N. benthamiana leaf proteins were extracted 3 days after inoculation, subjected to electrophoresis, and transferred to nitrocellulose membranes that were probed with polyclonal antisera to the RCNMV CP. Lane 1, mock-inoculated tissue; other lanes contain tissue inoculated with wild-type RCNMV (lane 2), RCNMV RNA-1 plus pHST2 (lane 3), and RCNMV RNA-1 plus pHST2-ΔBX (lane 4). (B) Predicted secondary structure within the 209-nt RCNMV RNA-2 region that displays trans-activator activity. Identical nucleotides between RCNMV and CRSV are shaded in gray; nucleotides that are identical to SCNMV are boxed. The vertical bracket identifies the core essential 34-nt trans-activator. The loop highlighted in black is complementary to an 8-nt element on RNA-1. The indicated RNA-2 sequence is located between nucleotides 746 and 916. Other sequence elements not displaying a high degree of conservation were omitted for clarity. (C) Northern (RNA) blot analysis of R1sGFP-pHST2 co-inoculations. Total RNA was extracted from N. clevelandii leaves 3 days after inoculation, subjected to electrophoresis, and transferred to nylon membranes that were subsequently probed with an sGFP-specific32P-labeled DNA. Premature termination products of viral replication are visible as a smear in lanes 2 to 4. Lanes are as follows: 1, mock; 2, R1sGFP + pHST2; 3, R1sGFP + pHST2-SL2; and 4, R1sGFP + RNA-2.

Two conserved stem-loop structures were predicted on the basis of alignments with the CRSV and SCNMV RNA-2 sequences, in conjunction with a computer-predicted secondary structure model of the 209-nt RCNMV RNA-2 sequence (12, 17) (Fig. 2B). Inserts from the regions containing each predicted stem loop were tested separately for their ability to trans-activate sGFP expression. Only the TBSV replicon containing the 5′-terminal 121 nt from this region of RNA-2 (18) induced sGFP expression from co-inoculated R1sGFP transcripts (Table 1). We next focused on the highly conserved 34-nt stem-loop sequence within this 121-nt region (Fig. 2B). The 34-nt RNA-2 sequence expressed from the TBSV replicon (19) in the genomic- but not the complementary-sense orientation was sufficient for sGFP expression at levels near those obtained by wild-type RNA-2 co-inoculation (Table 1). Northern blot hybridization analysis showed that the sgRNA containing the sGFP sequence is produced only when the 34-nt RNA-2 sequence is present (Fig. 2C). The terminal 21 nt of the stem-loop structure conserved among the Dianthoviruses (Fig. 2B) also elicited sGFP expression but at a much lower level. However, a 9-nt insert containing the 8 nt in the loop of the 34-nt element was not sufficient to induce sGFP expression (Table 1).

Sequence alignments revealed that the 8-nt loop from RNA-2 is complementary to an 8-nt sequence element on RNA-1 located 2 nt upstream from the start site of sgRNA synthesis and within a region broadly mapped to be the CP subgenomic promoter (20). To determine if these sequences formed base pairs, we mutated the 8-nt element on RNA-1 at three positions that would not alter the amino acid coding sequence of the p88 polymerase (21). Co-inoculation of transcripts from this mutant clone (R1sGFP-M) with TBSV transcripts containing the 34-nt RNA-2 trans-activator element did not elicit sGFP synthesis (Table 2). When compensatory mutations were incorporated into the loop sequence of the 34-nt RNA-2 element (pHST2-SL2-M) (19) to restore complementarity with the mutated RNA-1 element, sGFP expression was restored (Table 2).

Table 2

Expression of sGFP from compensatory mutations between the predicted loop sequence of the RCNMV RNA-2 trans-activator and the complementary RCNMV RNA-1 8-nt element in the CP subgenomic promoter. Mutated nucleotides are in bold typeface and underlined.

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These results establish that a 34-nt sequence, predicted to form a simple stem loop, trans-activates sgRNA synthesis on another RNA. Furthermore, mutation data support the conclusion that trans-activation is accomplished by base-pairing between the 8-nt loop region of the RNA-2 trans-activator and an 8-nt element within the CP subgenomic promoter on RNA-1. It is unclear whether the interaction of RNA-1 and RNA-2 requires proteins. Sequence elements nearly identical to the RCNMV RNA-1 8-nt element are also present at the same location in CRSV and SCNMV RNA-1, suggesting that this mechanism for sgRNA synthesis is conserved among the Dianthoviruses.

We propose the following model (22) for sgRNA synthesis (Fig. 3, A and B). During the early phase of replication, in the absence of high levels of RNA-2, the replicase produces full-length complementary strand copies of RNA-1. Later, during production of progeny RNAs, RNA-2 becomes abundant. The trans-activator element on RNA-2 binds to the 8-nt element on RNA-1, possibly mediated by interactions with viral or host proteins (or both), to produce a structure that sterically prevents the replicase from completing synthesis of full-length complementary strands of RNA-1. Because the 5′-terminal sequences of the genomic RNA-1 and the sgRNA are highly conserved (Fig. 3A), this truncated complementary strand RNA may serve as a template for replication to produce the genomic-sense sgRNA, which is the translational template for CP. The presence of a double-stranded RNA species in RCNMV-infected tissue, corresponding in size to this replicative intermediate, supports this model (5, 20). This mechanism for controlling the temporal expression of CP may have evolved to ensure that sufficient quantities of RNA-1 and RNA-2 are present before the commencement of virion assembly. This model for sgRNA synthesis differs from that proposed for most RNA plant viruses, as exemplified by brome mosaic virus (BMV). BMV sgRNA synthesis is initiated by binding of the viral polymerase complex to an internal sequence on the negative strand template followed by transcription of positive stranded mRNA for the CP (23). Our model is distinct because promoter specificity is not based solely on protein-RNA interactions but more on an 8-nt RNA-RNA interaction that occurs, probably with the aid of proteins, between two genomic-sense RNAs.

Figure 3

Proposed components and model of the RCNMV trans-activation mechanism. (A) Sequences involved in trans-activation. Gray shaded nucleotides represent conserved sequences between genomic RNA-1 and the sgRNA that are likely to be + strand promoters. The loop region of the 34-nt trans-activator is shown base-pairing with the complementary 8-nt element, 2 nt upstream from the sgRNA start site (right angle arrow) on RNA-1. (B) Model for the generation of sgRNA. Complementary strands are depicted as dashed lines.

  • * To whom correspondence should be addressed. E-mail: steve_lommel{at}ncsu.edu

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